PROJECT SUMMARY Most of the eukaryotic genome is transcribed, yielding a complex repertoire of protein-coding mRNAs and noncoding RNAs. Most long RNA polymerase II transcripts are thought to have a 5' cap and a 3' poly(A) tail, which protect the transcript from degradation as well as recruit the translation machinery. However, our recent work has revealed a number of abundant transcripts that are generated by non-canonical mechanisms and either lack a poly(A) tail (e.g. MALAT1) or have covalently linked ends (e.g. circular RNAs). MALAT1, which is commonly mis-regulated in many human cancers, ends in a triple helical structure that supports both RNA stability and translation. Likewise, thousands of protein-coding pre-mRNAs are non-canonically spliced to produce circular RNAs that are resistant to degradation by exonucleases, and some of these circular RNAs exceed the abundance of their associated linear mRNA by a factor of 10. Because these non-polyadenylated RNAs and circular RNAs are structurally distinct from canonical mRNAs, they are subjected to different biogenesis and post-transcriptional control mechanisms as well as likely bound by unique factors. However, little is currently known about how the fates of these non-canonical RNAs are controlled. We thus propose two complementary projects to address these gaps in knowledge. First, we will characterize how non- polyadenylated linear mRNAs, such as transcripts ending in a triple helix or viral-derived sequences, are stabilized and efficiently translated. We propose that just as poly(A) binding protein (PABP) binds the poly(A) tail and stem-loop binding protein (SLBP) binds the histone stem-loop to help recruit the translation machinery, there are likely unique factors that directly bind these less characterized 3' ends to regulate their expression. These novel mechanisms would thus allow these specific RNAs to be regulated by unique signaling cascades or only expressed in particular tissues. Using RNAi screening and binding assays, the key trans-acting factors and the mechanisms by which they recruit the ribosome to these RNAs will be identified. We additionally will identify other sequences that can functionally replace a poly(A) tail, thereby revealing new paradigms for how mRNA 3' ends are generated and regulated. Second, we will determine how circular RNA expression is controlled by cellular cues to impact cellular differentiation events. Most circular RNAs are expressed in a tissue-specific manner, yet the underlying mechanisms by which their expression levels are regulated are unknown. By profiling changes in circular RNA expression as cells differentiate, a set of transcripts that are dramatically altered will be identified. The mechanisms by which these circular RNAs are regulated and function will subsequently be characterized, revealing new insights into how these unexpected outputs of protein-coding genes control cell identity. In total, these innovative studies will reveal important insights into how non-canonical transcripts are post-transcriptionally regulated and function to impact both normal and diseased states.